The invention relates to a recombinant DNA molecule encoding a polypeptide having phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity as well as the coded polypeptide itself. In particular, the invention relates to a recombinant DNA molecule encoding a polypeptide having phytase activity and increased temperature stability and increased proteolytic stability of the enzyme activity, whereby the DNA sequence has been obtained by variation of the mature wild-type E. coli phytase sequence with defined amino acid positions being modified in comparison to the wild-type sequence or with the sequences having N- and/or C-terminal extensions, respectively. The invention further relates to a method for expressing the recombinant phytase as well as its use in the food and animal feed technologies.
Phytic acid or myoinositol-1,2,3,4,5,6-hexakisdihydrogenphosphate (abbreviated myoinositol hexakisphosphate) represents the main source of inositol and the principal storage form of phosphate in plant seeds. In the seeds of legumes about 70% of the phosphate content is present in the form of a mixture of potassium, magnesium and calcium salts of the phytic acid. Seeds, grains and legumes are important components of food and animal feed preparations, in particular of animal feed preparations; but also in human nutrition grains and legumes become more and more important.
The phosphate units of the phytic acid bind as complex bivalent and trivalent cations like metal ions, i.e. in terms of nutritional physiology important ions like calcium, iron, zinc and magnesium as well as the trace elements manganese, copper and molybdenum. Further, phytic acid also binds to a certain extent proteins via electrostatic interaction.
Often, phytic acid and its salts, the phytates, are not metabolised since they are not absorbed from the gastrointestinal tract; i.e. neither the therein contained phosphorus nor the chelated metal ions nor the bound proteins are available in terms of nutritional physiology.
Since phosphorus represents an essential element for the growth of all organisms food and animal feed have to be supplemented by anorganic phosphorus. Very often, ions like iron or calcium, which are essential in terms of nutritional physiology, have to be supplemented. Moreover, the value of each diet in terms of nutritional physiology is reduced since proteins are bound by phytic acid. Consequently, phytic acid is often described as anti-nutritional factor.
Further, due to the fact that the phytic acid is not metabolised, the phosphorus of the phytate is excreted via the gastrointestinal tract of the animals, leading to an undesired phosphate pollution of the environment, which might be the cause, for example, for eutrophication of water bodies, and to excessive growth of algae.
Phytic acid or phytate (unless indicated otherwise, these terms are used in the following as synonyms) can be metabolised by phytases. Phytases catalyse the hydrolysis of phytate to myoinositol and/or mono-, di-, tri-, tetra- and/or pentaphosphate as well as anorganic phosphate. Two different forms of microbial phytases are distinguished from each other: 1) 3-phytase/myoinositiolhexaphosphate-3-phosphohydrolase, EC 3.1.3.8; 2) 6-phytase/myoinositolhexaphosphate-6-phosphohydrolase, EC 3.1.3.26. The 3-phytase preferably hydrolyses first the ester bond in position 3, the 6-phytase preferably first the ester bond in position 6. Phytic acid containing plant seeds contain endogenous phytase enzymes. When ingesting the same, the phytates in food or animal feed are theoretically hydrolysable by endogenous plant phytases, phytases from the intestinal flora and phytases from the intestinal mucosa. In practice however, the potency of hydrolysis of the endogenous plant phytases and the phytases found in the intestine, if existing, is by far insufficient in order to assure significantly the bioavailability of the phosphorus bound in the phytates. Thus, exogenous phytases are often added to food and animal feed.
Phytates can be produced by plants and by microorganisms. Among the microorganisms phytase producing bacteria as well as phytase producing fungi and yeasts are known.
The naturally occurring phytase producers, however, have the disadvantage that phytase is generated only in certain amounts and with defined characteristics. As described hereinbefore, though, there exists an increased demand for phytase in particular in the food and animal feed industries.
Although an increased demand for phytase in the food and animal feed industries does exist and the use of phytase might be advantageous only a few of the known phytases have found a broad acceptance in the food and animal feed industries. Typical concerns relate to the comparatively high production costs and/or the poor stability, or activity of the enzyme in the desired application environment. Moreover, such an enzyme has to fulfil certain criteria in order to be industrially used. Those comprise a high specific total activity, a low pH-optimum, resistance against gastrointestinal proteases as well as temperature stability, or thermostability. The temperature stability is an important prerequisite for a successful industrial application since, for example, enzymes are exposed to temperatures between 60° C. and 95° C. in pelletising processes.
All known microbial phytases unfold at temperatures between 56° C. and 78° C. (Lehman et al., 2000), whereby they lose their activity. Therefore there exists a particular demand for phytases which possess a technologically sufficient activity also at higher temperatures, or which are not inactivated.
Thus, an object of the present invention is to provide a polypeptide having phytase activity which exhibits an increased thermostability, or which possesses a technologically sufficient activity at higher temperatures. Moreover, the polypeptide having phytase activity should also possess an increased proteolytic stability.
It is desired that the polypeptide be produced economically. In particular, the phytase should have a higher thermostability than the wild-type enzyme. Moreover, the phytase should keep the essential characteristics of the natural E. coli-wild-type phytase but feature an improved thermostability. Among the essential characteristics of the natural wild-type phytase are in particular the capability of improving the availability of phosphate in vivo and in vitro by its activity as phytase as well as phosphatase, its pH-optimum in an acidic environment with high residual activity in a highly acidic environment as well as the applicability as additive for baking.
An object of the present invention is further to provide a gene for a polypeptide having phytase activity and having increased thermostability as well as increased proteolytic stability. It is desired that the polypeptide be produced economically and in a cost-effective way. In particular, the expression of the polypeptide in eukaryotic microorganisms should lead to a polypeptide with increased thermostability compared to the similarly produced wild-type phytase. Further are to be provided: the DNA sequences encoding the polypeptide, corresponding DNA constructs and vectors as well as a source for the recombinant enzyme which is suitable for the commercial use for food and animal feed in industrial processes and compositions containing the enzyme according to the invention.
It was surprisingly found that at certain positions of the E. coli wild-type phytase sequence mutations lead to an intrinsic improvement of the thermostability, or temperature stability as well as to an improvement of the proteolytic stability of the protein phytase without affecting adversely the other effects and essential characteristics of the wild-type E. coli phytase.
It was surprisingly found that a mutation in position 74 (K74D) of the amino acid sequence and/or a combination of mutations in positions 139 (N139R) and 142 (D142E) and/or a combination of mutations in positions 145 (L145I) and 198 (L198I) and/or a mutation in position 200 (V200P) of the wild-type phytase of E. coli as well as combinations of these mutations lead to an improved thermostability of the protein phytase without affecting the advantageous effects and essential characteristics of the wild-type E. coli phytase. It was further found that the extension of E. coli phytase by sequences of the acidic phosphatase of Aspergillus niger var. awamori at the N-terminal or C-terminal end or at the N-terminal and C-terminal ends, also in combination with the afore-mentioned mutations leads to an improvement of the thermostability and the proteolytic stability of the enzyme.
The invention thus relates to a recombinant DNA molecule encoding a polypeptide having phytase activity after being expressed in a prokaryotic or eukaryotic host cell, whereby the recombinant DNA molecule comprises a DNA sequence selected from                a) DNA sequences encoding a polypeptide that has phytase activity and is obtained by varying of the mature wild-type E. coli phytase sequence, the variation being selected from among                    i) the mutation lysine→aspartic acid in position 74 (K74D), and/or            ii) a combination of the mutations asparagine→arginine in position 139 (N139R) and aspartic acid→glutamic acid in position 142 (D142E), and/or            iii) a combination of the mutations leucine→isoleucine in position 145 (L145I) and leucine→isoleucine in position 198 (L198I), and/or            iv) a mutation valine proline in position 200 (V200P), and/or            v) an N-terminal or C-terminal or an N-terminal and C-terminal addition of a sequence section of the acidic phosphatase of Aspergillus niger var. awamori or the phytase of Aspergillus niger,                         b) DNA sequences that are 70 to 100 percent homologous to the sequences listed under a)        c) DNA sequences which are related to the sequences listed under a) and b) because of the degeneration of the genetic code,whereby the recombinant DNA molecule, when expressed in a suitable host cell, has an increased temperature and protease stability of the enzyme activity of the protein coded in said manner, whereby the variations iii) and iv) are provided only in combination with a variation i), ii) and v) as well as the polypeptide sequences coded by said DNA.        
Several phytases from E. coli are described in literature, e.g. the appAgene from E. coli K-12 which codes a phytase (Dassa et al., J. Bacteriol. 172:5497-5500 (1990)). This gene codes for a periplasmatic enzyme which comprises acidic phosphatase activity as well as phytase activity (cf Greiner et al., Arch. Biochim, Biophys. 303:107-113 (1993)). Natural mutants of this enzyme are known (cf, for example, Rodriguez et al., Biochem. Biophys. Res. Comm. 257:117-123 (1999)). Genetically modified mutants of E. coli phytase have also been described which lead to an increased temperature stability and/or an increased specific activity. Rodriguez et al. (Arch. Biochem. Biophys. 382:105-112 (2000)) expressed wild-type AppA and several mutants created by site-specific mutagenesis in Pichia pastoris in order to test the effect of N-glycolysation on the temperature stability of the AppA protein. Although the glycolysation has not been intensified a mutant has been more active at a pH between 3.5 and 5.5 and has shown more activity after the heating treatment than the wild type protein produced in P. pastoris. 
The patent family based on WO 03/057248 comprises the patent applications US 2003/0170293 A1 and US 2003/0157646 A1. Therein, the microbial production of a thermotolerant phytase for animal feed is described. The mutant (Nov9X) of the E. coli strain B phytase (appA) is expressed in E. coli, Pichia pastoris, and Schizosaccaromyces pombe. The mutant Nov9X comprises 8 amino acid mutations compared to the wild-type enzyme. The mutant has a better thermostability in liquid at 70° C. compared to the wild-type enzyme. The host in which the enzyme is produced has also an influence on its thermostability, as implied by the same work group in US patent application US 2003/0157646 A1. The method of counting the amino acid position is by two positions higher for NOV9X than in the present invention (W48 NOV9X corresponds to W46 in the present invention).
In the patent applications WO 02/095003 and WO 2004/015084, a number of point mutations of E. coli phytase are described; none of them, however, leads to an increase of the thermostability. Compared to the present invention, the counting in WO 2004/015084 is by 30 amino acid positions higher.
Publication document DE 10 2004 050 410 also describes E. coli phytase mutants, with the aim, however, to increase the secretion efficiency during production in filamentous fungi. No information about the increase of the thermostability of the mutants is given in said document.
Further, document Garrett et al.; Applied Environ. Microbiol., 2004, 70 (5), 3041-3046, describes mutants of E. coli phytase with an increased thermal and gastrointestinal stability.
It is practically impossible to predict the effect of one or several mutants on the characteristics of the enzyme activity under conditions of higher temperatures. Higher temperatures generally lead to a denaturation, or to an unfolding of the secondary and tertiary structure of the protein.
The temperature stability or thermostability of proteins depends on a number of interactions. The conformation of proteins is maintained by a huge number of weak interactions. The stabilisation can comprise all hierarchical levels of the protein structure: local packing of the polypeptide chain, secondary and supersecondary structural elements, domains and subunits of a multimeric protein. There are various reasons which are described concerning the increased temperature stability of a protein, whereby the most common are: (a) the association of ion pairs within and/or between subunits; (b) improved packing of the hydrophobic core (van-der-Waals interactions); (c) additional networks of hydrogen bridges; (d) increased tendency towards secondary structure building; (e) increased dipole stabilisation within the helix; (f) an increased polar surface; (g) reduced number and a reduced total volume of cavities; (h) reduction of conformational stress (loop stabilisation) and (i) resistance against chemical modification (oxidation of methionine residues and/or deamination of aspartic and glutamic residues).
The prior art furnishes no indications as to how the E. coli wild-type phytase sequence is preferably to be altered in order to gain an increase of the thermostability. Moreover, the prior art furnishes no indications with respect to the afore-mentioned mutations in the E. coli phytase sequence.
In particular, the prior art furnishes no indications that a mutation in position 74 (K74D) of the amino acid sequence or a combination of mutations in positions 139 (N139R) and 142 (D142E) or a combination of mutations in positions 145 (L145I) and 198 (L198I) or a mutation in position 200 (V200P) of wild-type phytase of E. coli or combinations of these mutations lead to an improved thermostability of the protein phytase without affecting the advantageous effects and essential characteristics of the wild-type E. coli phytase. Moreover, the prior art furnishes no indications that the supplementation of the E. coli phytase sequence by sequences of the acidic phosphatase of Aspergillus niger var. awamori at the N-terminal or C-terminal or at the N-terminal and C-terminal, also in combination with the afore-mentioned mutations, lead to an improvement of the thermostability of the enzyme.